Several procedures for the isolation of DNA or RNA from biological samples have been published. One of the earliest procedures for isolation of bacterial DNA utilized a precipitation with alkyltrimethylammonium bromides; see Jones, Biochim. Biophys. Acta (1953) 10:607-612; and Jones, Nature (1963), 199: 280-282. In the Jones procedure, the alkyltrimethylammonium bromides also precipitate other components, the salts must be completely removed, and a subsequent series of extractions and precipitations are required to obtain the purified DNA, a procedure which takes 1-2 days. No complex biological samples were used.
A later procedure for isolation of DNA from microorganisms was first described in Marmur, J. Mol. Biol. (1961) 3:208-218. The Marmur procedure also involves a series of extractions and precipitations utilizing the caustic reagents phenol and chloroform. Both the Marmur and the Jones procedures use ethanol as a precipitating agent. The Marmur procedure is still considered the state of the art, but requires 1-2 days to perform. Both methods use harsh organic solvents, which additionally make them clinically unacceptable.
From some biological fluids such as urine or blood, other direct and rapid preparation methods are known, such as direct lysing and spotting. For example, see Gillespie, et al., BioTechniques Nov./Dec. 174-192 (1983) and Barker et al., Science 231:1434-1436 (1986). Also see general techniques described in Nucleic Acid Hybridization: A Practical Approach, B. Hanes and S. Higgins, eds., IRL Press, Washingon (1985). As the authors themselves discuss, however, the methods cannot be used directly for clinically relevant sample sizes, for even in these relatively simple samples interference from components other than nucleic acids become unacceptable.
In addition to these extraction/precipitation purification methods, chromatographic column separation procedures have been proposed using several specially designed column packing materials, viz. the "NENSORB 20" cartridges sold by DuPont NEN Products, Boston, Mass., which function primarily by reverse phase adsorption and are not recommended for biological samples, the "NUCLEOGEN" DEAE weak anion exchange materials for HPLC sold by Machery-Nagel, Duren, West Germany; and strong anion exchange materials used for HPLC, such as Whatman Partisil SAX. However, the chromatographic column systems heretofore used are lengthy, require HPLC instrumentation that mandate high pressure to obtain adequate separation and are incapable of isolating nucleic acids from complex biological samples containing many components other than nucleic acids, such as those prepared from feces, blood or other bodily fluids or tissues. Adding such samples to these column systems would cause unavoidable degradation, resolution loss and blockage of the column. As such, in order to make these systems practical for adequate isolation of nucleic acids, significant sample pretreatment is required and mechanical backpressures necessitate the use of sophisticated pumping and delivery systems.
State of the art isolations of DNA/RNA continue to employ a variation of the twenty year old method described by Marmur, which utilizes many tedious and time consuming steps, requires numerous treatments with unacceptable organic solvents and does not provide adequately isolated nucleic acids needed for hybridization assays in a clinical laboratory. Contaminates in the biological samples interfere with fixing DNA/RNA hybridization and detection. As a consequence, to provide useful clinical hybridization assays, there exists a need for rapid methods to obtain hybridization nucleic acids from biological samples. Currently, such a system does not exist. Such methods must be rapid--(1) minutes rather than hours or days; (2) be efficient--can recover sufficient RNA or DNA to detect hybridization; (3) require little or no sample pretreatment; (4) not require expensive instrumentation or pumps; (5) not use organic solvents or vigorous conditions; (6) be essentially universal in nature--can be used for any length RNA or DNA, single or double-stranded; and (7) result in high purity DNA or RNA, thus eliminating background contaminants from the biological material. The present invention fills that need with a novel method combining rapid lysis of samples with subsequent isolation of nucleic acids contained in highly contaminated biological samples by a simplified anion exchange separation.
A laboratory procedure is known in which DNA can be recovered from agarose gels by electrophoresis which separates the DNA from sulfated mucopolysaccharides present in the sample. This procedure recognizes that the mucopolysaccharides usually extract from the sample together with the DNA, and uses this special electrophoresis procedure to separate these components. Following the separation of the DNA from the mucopolysaccharides by electrophoresis, and the DNA fraction is cut out and the DNA electroeluted it is proposed to further purify the gel by passing it through "DEAE-Sephacel". In this procedure the DNA applied to the column is washed with 0.3M aqueous NaCl, and thereafter the DNA is eluted with 0.6M aqueous NaCl. The eluant is extracted twice with phenol, once with phenol/chloroform, and once with chloroform. The DNA is then recovered from the purified eluant by precipitation with ethanol. See Maniatis, et al., "Molecular Cloning" (1982), pages 164-166. Although the above described procedure does separate mucopolysaccharides from the desired DNA, the method is laborious. It involves electrophoresis elution, column separation, and phenol chloroform extraction.